Idiopathic (primary) pulmonary arterial hypertension (IPAH), a subgroup of the vascular injury-induced forms of pulmonary arterial hypertension (PAH), is a rare disorder associated with severe morbidity and high mortality rates. There are no routine screening tests or validated markers of disease activity in IPAH, or the broader group of PAH. Therefore, patients usually present at advanced stages of disease. The pathogenesis of IPAH and other forms of PAH remain unclear. Current thinking focuses on a two-hit hypothesis: 1) genetic susceptibility, and 2) a triggering stimulus that initiates pulmonary vascular injury, resulting in endothelial cell dysfunction. Endothelial cells are normally shed into the circulation and are a valuable source of clinical material for studying diseases characterized by endothelial cell dysfunction. Unfortunately, no clear methodology exists for isolating clinically relevant numbers of circulating endothelial cells (CECs). In the bench phase of the project we were using flow cytometry to develop a methodology for isolating clinically relevant numbers of viable CECs from healthy volunteers and patients with PAH. We hypothesize that CECs and/or peripheral blood mononuclear cells (PBMC) can be used to define a subset of differentially regulated biomarkers in IPAH and other forms of PAH that may lead to earlier diagnosis and better methods for measuring responses to therapy. We also hope to identify novel targets for future therapeutic interventions. In the clinical phase of the project, we recruited healthy volunteers and patients with IPAH and other forms of PAH (vascular injury induced pulmonary hypertension). Peripheral blood specimens were obtained for CECs and PBMCs for microarrays; the remaining plasma was saved for future application to cultured microvascular cells. A subset of subjects underwent right heart catheterization to assess pulmonary pressures and to obtain pulmonary blood specimens. We started actively enrolling into the protocol in June 2006. We enrolled 31 individuals prior to closing the protocol to enrollment in 2009. Preliminary data suggested that there was no trend towards CEC enrichment in pulmonary vein blood compared to peripheral blood (PB) for both the healthy volunteers (4.4 CEC/ml vs. 4.8 CEC/ml) and the PAH patients (2.4 CEC/ml vs. 3.0 CEC/ml). There was a trend towards CEC enrichment in pulmonary artery blood compared to PB for both the healthy volunteers (13.8 CEC/ml vs. 4.8 CEC/ml) and the PAH patients (3.3 CEC/ml vs. 3.0 CEC/ml). In 2010 and 2011, total RNA was processed from PBMCs for genome-wide expression analysis. An abstract based on the PBMC differential gene expression patterns in PAH was presented at the Annual American Thoracic Society Meeting in 2011. These patterns reflected both treatment related signatures and underlying disease pathophysiology. In addition to completing expression profiling of PBMCs, plasma samples from healthy volunteers and patients with PAH were processed for application to cultured microvascular endothelial cells. In 2011 to 2013, using PBMC expression profiles from 10 PAH subjects with 10 age, gender and race matched healthy control subjects we identified over 230 differentially regulated genes at a 20% false discovery rate. Ingenuity Pathway Analysis identified gene signatures for inflammation, cell-to-cell signaling and interaction, cytoskeletal rearrangement, cellular movement, hemostasis and cell death. In vitro data from our collaborating laboratory demonstrates that spironolactone suppresses phorbol 12-myristate 13-acetate-induced (PMA; an AP-1 activator) inflammatory gene transcription in primary human PAECs. In order to explore the effect of spironolactone on PAH-associated vascular inflammation we conducted a promoter level analysis of the up-regulated genes we identified in subjects with PAH. Biobase ExPlain and Transfac bioinformatics software identified activator protein-1 (AP-1) as a key transcriptional regulator. Experiments using PBMCs isolated from healthy subjects and stimulated with PMA demonstrate that spironolactone suppresses these AP-1 inducible, PAH-associated genes in a dose-dependent manner. Similarly in PBMCs from healthy subjects and PAH subjects, spironolactone strongly suppressed the basal expression of genes that had been up regulated in the PBMCs of patients with PAH. In addition in 2011 -12 we continued to develop a bioassay assessing global transcriptomic changes induced by plasma from PAH subjects compared to healthy controls using Affymetrix oligonucleotide microarrays. Exposure of human PAECs to plasma from 5 PAH subjects compared to 5 age, gender and race matched controls, identified over 300 differentially expressed transcripts at a 10% false discovery rate. In additional work done in 2012-13, we explored the gene expression changes in cultured human PAECs induced by plasma from PAH subjects and found that 20% of this signature overlapped with the gene expression changes induced following bone morphogeneic protein receptor-2 (BMPR2) gene silencing in PAECs. Importantly, more than 90% of this overlap was directionally discordant, suggesting circulating factors may work to counter-regulate genotypic and phenotypic abnormalities that drive PAH. Future experiments will utilize stored plasma currently available from PAH patients and healthy controls to examine the effects of circulating mediators on gene expression in BMPR2-deficient PAECs. In 2013-14, we have continued to actively investigate the mechanisms that mediate the anti-inflammatory effects of spironolactone using molecular techniques such as overexpression vectors and AP-1 promoter based reporter assays. Furthermore, in order to expand upon our expression findings in circulating mononuclear cells, we have collected data from all of the other published human PAH PBMC genome-wide expression profiling studies for subsequent meta-analysis. Once data annotation and aggregation is completed, we plan to compare PBMC gene expression of IPAH and disease-associated PAH to healthy controls as well as to each other. This protocol remains open to continue bioinformatic analyses of the gene expression data, completing downstream in vitro work as well as finishing a meta-analysis of the published PAH PBMC expression profiling studies.